Harris, Peter C

Abstract DNA damage response (DDR) is an important signaling-transduction network that promotes the repair of DNA lesions which can induce and/or support diseases. However, the mechanisms involved in its regulation are not fully understood. Recent studies suggest that the peroxiredoxin 5 (Prdx5) enzyme, which detoxifies reactive oxygen species, is associated to genomic instability and signal transduction. Its role in the regulation of DDR, however, is not well characterized. In this study, we demonstrate a role of Prdx5 in the regulation of the DDR signaling pathway. Knockdown of Prdx5 resulted in DNA damage manifested by the induction of phosphorylated histone H2AX (γ-H2AX) and p53-binding protein 1 (53BP1). We show that Prdx5 regulates DDR through (1) polo-like kinase 1 (Plk1) mediated phosphorylation of ataxia telangiectasia mutated (ATM) kinase to further trigger downstream mediators Chek1 and Chek2; (2) the increase of the acetylation of p53 at lysine 382, stabilizing p53 in the nucleus and enhancing transcription and (3) the induction of autophagy, which regulates the recycling of molecules involved in DDR. We identified Sirt2 as a novel deacetylase of p53 at lysine 382, and Sirt2 regulated the acetylation status of p53 at lysine 382 in a Prdx5-dependent manner. Furthermore, we found that exogenous expression of Prdx5 decreased DNA damage and the activation of ATM in Pkd1 mutant renal epithelial cells, suggesting that Prdx5 may play a protective role from DNA damage in cystic renal epithelial cells. This study identified a novel mechanism of Prdx5 in the regulation of DDR through the ATM/p53/Sirt2 signaling cascade.

Significance Statement The only treatment approved for PKD inhibits production of cAMP, the main PKA activator. It is only partially effective, likely because side effects restrict dosing and because other sources of cAMP and mechanisms of cAMP-independent PKA activation exist. Which PKA isozyme(s) promotes PKD is uncertain and selective PKA inhibitors usable in vivo have not been available. Experiments in a mouse model show PKA-I is the main PKA isozyme promoting cystogenesis and that constitutive PKA-I downregulation and a novel, highly selective PKA inhibitor ameliorate PKD. The dose of PKA inhibitor used had no detectable adverse effects. This information provides a strong rationale for a strategy that may be more effective, or substantially increase the efficacy of the currently approved treatment. Background Upregulation of cAMP-dependent and cAMP-independent PKA signaling is thought to promote cystogenesis in polycystic kidney disease (PKD). PKA-I regulatory subunit RI α is increased in kidneys of orthologous mouse models. Kidney-specific knockout of RI α upregulates PKA activity, induces cystic disease in wild-type mice, and aggravates it in Pkd1 RC/RC mice. Methods PKA-I activation or inhibition was compared with EPAC activation or PKA-II inhibition using Pkd1 RC/RC metanephric organ cultures. The effect of constitutive PKA (preferentially PKA-I) downregulation in vivo was ascertained by kidney-specific expression of a dominant negative RIαB allele in Pkd1 RC/RC mice obtained by crossing Prkar1α R1αB/WT , Pkd1RC/RC , and Pkhd1- Cre mice (C57BL/6 background). The effect of pharmacologic PKA inhibition using a novel, selective PRKACA inhibitor (BLU2864) was tested in mIMCD3 3D cultures, metanephric organ cultures, and Pkd1RC/RC mice on a C57BL/6 × 129S6/Sv F1 background. Mice were sacrificed at 16 weeks of age. Results PKA-I activation promoted and inhibition prevented ex vivo P-Ser133 CREB expression and cystogenesis. EPAC activation or PKA-II inhibition had no or only minor effects. BLU2864 inhibited in vitro mIMCD3 cystogenesis and ex vivo P-Ser133 CREB expression and cystogenesis. Genetic downregulation of PKA activity and BLU2864 directly and/or indirectly inhibited many pro-proliferative pathways and were both protective in vivo . BLU2864 had no detectable on- or off-target adverse effects. Conclusions PKA-I is the main PKA isozyme promoting cystogenesis. Direct PKA inhibition may be an effective strategy to treat PKD and other conditions where PKA signaling is upregulated. By acting directly on PKA, the inhibition may be more effective than or substantially increase the efficacy of treatments that only affect PKA activity by lowering cAMP.

Background and objectives The progression of polycystic liver disease is not well understood. The purpose of the study is to evaluate the associations of polycystic liver progression with other disease progression variables and classify liver progression on the basis of patient's age, height-adjusted liver cystic volume, and height-adjusted liver volume. Design, setting, participants, & measurements Prospective longitudinal magnetic resonance images from 670 patients with early autosomal dominant polycystic kidney disease for up to 14 years of follow-up were evaluated to measure height-adjusted liver cystic volume and height-adjusted liver volume. Among them, 245 patients with liver cyst volume >50 ml at baseline were included in the longitudinal analysis. Linear mixed models on log-transformed height-adjusted liver cystic volume and height-adjusted liver volume were fitted to approximate mean annual rate of change for each outcome. The association of sex, body mass index, genotype, baseline height-adjusted total kidney volume, and Mayo imaging class was assessed. We calculated height-adjusted liver cystic volume ranges for each specific age and divided them into five classes on the basis of annual percentage increase in height-adjusted liver cystic volume. Results The mean annual growth rate of height-adjusted liver cystic volume was 12% (95% confidence interval, 11.1% to 13.1%; P <0.001), whereas that for height-adjusted liver volume was 2% (95% confidence interval, 1.9% to 2.6%; P <0.001). Women had higher baseline height-adjusted liver cystic volume than men, but men had higher height-adjusted liver cystic volume growth rate than women by 2% (95% confidence interval, 0.4% to 4.5%; P =0.02). Whereas the height-adjusted liver cystic volume growth rate decreased in women after menopause, no decrease was observed in men at any age. Body mass index, genotype, and baseline height-adjusted total kidney volume were not associated with the growth rate of height-adjusted liver cystic volume or height-adjusted liver volume. According to the height-adjusted liver cystic volume growth rate, patients were classified into five classes (number of women, men in each class): A (24, six); B (44, 13); C (43, 48); D (28, 17); and E (13, nine). Conclusions Compared with height-adjusted liver volume, the use of height-adjusted liver cystic volume showed greater separations in volumetric progression of polycystic liver disease. Similar to the Mayo imaging classification for the kidney, the progression of polycystic liver disease may be categorized on the basis of patient’s age and height-adjusted liver cystic volume.

Key Points Asymptomatic pyuria is associated with kidney failure and faster kidney function decline irrespective of the ADPKD gene and cystic growth. The eGFR decline occurred after detection of asymptomatic pyuria without significant changes in the rate of total kidney volume growth. This study supports the use of asymptomatic pyuria as an enriching prognostic biomarker to predict faster disease progression. Background Autosomal dominant polycystic kidney disease (ADPKD) has phenotypic variability only partially explained by established biomarkers that do not readily assess pathologically important factors of inflammation and kidney fibrosis. We evaluated asymptomatic pyuria (AP), a surrogate marker of inflammation, as a biomarker for disease progression. Methods We performed a retrospective cohort study of adult patients with ADPKD. Patients were divided into AP and no pyuria (NP) groups. We evaluated the effect of pyuria on kidney function and kidney volume. Longitudinal models evaluating kidney function and kidney volume rate of change with respect to incidences of AP were created. Results There were 687 included patients (347 AP, 340 NP). The AP group had more women (65% versus 49%). Median ages at kidney failure were 86 and 80 years in the NP and AP groups (log rank, P =0.49), respectively, for patients in Mayo Imaging Class (MIC) 1A–1B as compared with 59 and 55 years for patients in MIC 1C–1D–1E (log rank, P =0.02), respectively. Compared with the NP group, the rate of kidney function (ml/min per 1.73 m 2 per year) decline shifted significantly after detection of AP in the models, including all patients (−1.48; P <0.001), patients in MIC 1A–1B (−1.79; P <0.001), patients in MIC 1C–1D–1E (−1.18; P <0.001), and patients with PKD1 (−1.04; P <0.001). Models evaluating kidney volume rate of growth showed no change after incidence of AP as compared with the NP group. Conclusions AP is associated with kidney failure and faster kidney function decline irrespective of the ADPKD gene, cystic burden, and cystic growth. These results support AP as an enriching prognostic biomarker for the rate of disease progression.

Dent disease is a rare X-linked renal tubulopathy due to CLCN5 and OCRL (DD2) mutations. OCRL mutations also cause Lowe syndrome (LS) involving the eyes, brain and kidney. DD2 is frequently described as a mild form of LS because some patients may present with extra-renal symptoms (ESs). Since DD2 is a rare disease and there are a low number of reported cases, it is still unclear whether it has a clinical picture distinct from LS. We retrospectively analyzed the phenotype and genotype of our cohort of 35 DD2 males and reviewed all published DD2 cases. We analyzed the distribution of mutations along the OCRL gene and evaluated the type and frequency of ES according to the type of mutation and localization in OCRL protein domains. The frequency of patients with at least one ES was 39%. Muscle findings are the most common ES (52%), while ocular findings are less common (11%). Analysis of the distribution of mutations revealed (1) truncating mutations map in the PH and linker domain, while missense mutations map in the 5-phosphatase domain, and only occasionally in the ASH-RhoGAP module; (2) five OCRL mutations cause both DD2 and LS phenotypes; (3) codon 318 is a DD2 mutational hot spot; (4) a correlation was found between the presence of ES and the position of the mutations along OCRL domains. DD2 is distinct from LS. The mutation site and the mutation type largely determine the DD2 phenotype.

HYLS1 regulates the ciliary phosphoinositide and hedgehog pathways, implicating a novel pathogenic mechanism for HLS syndrome.

Abstract Background Autosomal dominant polycystic kidney disease (ADPKD) is characterized by the progressive growth of cysts and a decline of renal function. The clinical feasibility of the number of potential disease-modifying drugs is limited by systemic adverse effects. We hypothesize that megalin, a multiligand endocytic receptor expressed in the proximal tubule, may be used to facilitate drug uptake into cysts, thereby allowing for greater efficacy and fewer side effects. Methods The cyst expression of various tubular markers, including megalin and aquaporin 2 (AQP2), was analysed by immunohistochemistry (IHC) of kidney sections from the ADPKD mouse model (PKD1RC/RC) at different post-natal ages. The endocytic function of megalin in cysts was examined by IHC of kidney tissue from mice injected with the megalin ligand aprotinin. Results Cyst lining epithelial cells expressing megalin were observed at all ages; however, the proportion decreased with age. Concomitantly, an increasing proportion of cysts revealed expression of AQP2, partial expression of megalin and/or AQP2 or no expression of the examined markers. Endocytic uptake of aprotinin was evident in megalin-positive cysts, but only in those that remained connected to the renal tubular system. Conclusions Megalin-expressing cysts were observed at all ages, but the proportion decreased with age, possibly due to a switch in tubular origin, a merging of cysts of different tubular origin and/or a change in the expression pattern of cyst lining cells. Megalin expressed in cysts was functional, suggesting that megalin-mediated endocytosis is a potential mechanism for drug targeting in ADPKD if initiated early in the disease.

Significance Statement Zebrafish embryos are well recognized for their value in studying polycystic kidney disease, but they cannot fully recapitulate the pathogenesis of the disease. The establishment of tmem67 e3/e3 mutants provides the first adult zebrafish model for cystogenesis. The model exhibits progressive renal cysts and enables the discernment of compensational effects from pathogenic events. Development of a tissue-clearing, whole-mount imaging protocol allows the analysis of renal cysts and cilia abnormality at a single-nephron resolution. Evidence also indicates potential benefit of mammalian target of rapamycin inhibition in tmem67 -based cystogenesis. Combining powerful genetics and phenotyping tools, this novel adult zebrafish model shall facilitate mechanistic studies and therapeutic development for cystic diseases. Background Although zebrafish embryos have been used to study ciliogenesis and model polycystic kidney disease (PKD), adult zebrafish remain unexplored. Methods Transcription activator-like effector nucleases (TALEN) technology was used to generate mutant for tmem67 , the homolog of the mammalian causative gene for Meckel syndrome type 3 (MKS3). Classic 2D and optical-clearing 3D imaging of an isolated adult zebrafish kidney were used to examine cystic and ciliary phenotypes. A hypomorphic mtor strain or rapamycin was used to inhibit mTOR activity. Results Adult tmem67 zebrafish developed progressive mesonephric cysts that share conserved features of mammalian cystogenesis, including a switch of cyst origin with age and an increase in proliferation of cyst-lining epithelial cells. The mutants had shorter and fewer distal single cilia and greater numbers of multiciliated cells (MCCs). Absence of a single cilium preceded cystogenesis, and expansion of MCCs occurred after pronephric cyst formation and was inversely correlated with the severity of renal cysts in young adult zebrafish, suggesting a primary defect and an adaptive action, respectively. Finally, the mutants exhibited hyperactive mTOR signaling. mTOR inhibition ameliorated renal cysts in both the embryonic and adult zebrafish models; however, it only rescued ciliary abnormalities in the adult mutants. Conclusions Adult zebrafish tmem67 mutants offer a new vertebrate model for renal cystic diseases, in which cilia morphology can be analyzed at a single-nephron resolution and mTOR inhibition proves to be a candidate therapeutic strategy.

Abstract Background Primary hyperoxaluria (PH) type 3 (PH3) is caused by mutations in the hydroxy-oxo-glutarate aldolase 1 gene. PH3 patients often present with recurrent urinary stone disease in the first decade of life, but prior reports suggested PH3 may have a milder phenotype in adults. This study characterized clinical manifestations of PH3 across the decades of life in comparison with PH1 and PH2. Methods Clinical information was obtained from the Rare Kidney Stone Consortium PH Registry (PH1, n = 384; PH2, n = 51; PH3, n = 62). Results PH3 patients presented with symptoms at a median of 2.7 years old compared with PH1 (4.9 years) and PH2 (5.7 years) (P = 0.14). Nephrocalcinosis was present at diagnosis in 4 (7%) PH3 patients, while 55 (89%) had stones. Median urine oxalate excretion was lowest in PH3 patients compared with PH1 and PH2 (1.1 versus 1.6 and 1.5 mmol/day/1.73 m2, respectively, P &lt; 0.001) while urine calcium was highest in PH3 (112 versus 51 and 98 mg/day/1.73 m2 in PH1 and PH2, respectively, P &lt; 0.001). Stone events per decade of life were similar across the age span and the three PH types. At 40 years of age, 97% of PH3 patients had not progressed to end-stage kidney disease compared with 36% PH1 and 66% PH2 patients. Conclusions Patients with all forms of PH experience lifelong stone events, often beginning in childhood. Kidney failure is common in PH1 but rare in PH3. Longer-term follow-up of larger cohorts will be important for a more complete understanding of the PH3 phenotype.

ABSTRACT Autosomal dominant polycystic kidney disease (ADPKD) is the most common inherited cause of chronic kidney disease (CKD) and leads to a specific type of bone disease. The primary cilium is a major cellular organelle implicated in the pathophysiology of ADPKD caused by mutations in polycystin‐1 ( PKD1 ) and polycystin‐2 ( PKD2 ). In this study, for the first time, cilia were characterized in primary preosteoblasts isolated from patients with ADPKD. All patients with ADPKD had low bone turnover and primary osteoblasts were also obtained from patients with non‐ADPKD CKD with low bone turnover. Image‐based immunofluorescence assays analyzed cilia using standard markers, pericentrin, and acetylated‐α‐tubulin, where cilia induction and elongation were chosen as relevant endpoints for these initial investigations. Osteoblastic activity was examined by measuring alkaline phosphatase levels and mineralized matrix deposition rates. It was found that primary cilia can be visualized in patient‐derived osteoblasts and respond to elongation treatments. Compared with control cells, ADPKD osteoblasts displayed abnormal cilia elongation that was significantly more responsive in cells with PKD2 nontruncating mutations and PKD1 mutations. In contrast, non‐ADPKD CKD osteoblasts were unresponsive and had shorter cilia. Finally, ADPKD osteoblasts showed increased rates of mineralized matrix deposition compared with non‐ADPKD CKD. This work represents the first study of cilia in primary human‐derived osteoblasts from patients with CKD and patients with ADPKD who have normal kidney function, offering new insights as bone disease phenotypes are not well recapitulated in animal models. These data support a model whereby altered cilia occurs in PKD‐ mutated osteoblasts, and that ADPKD‐related defects in bone cell activity and mineralization are distinct from adynamic bone disease from patients with non‐ADPKD CKD. © 2021 The Authors. JBMR Plus published by Wiley Periodicals LLC. on behalf of American Society for Bone and Mineral Research.